PROJECT SUMMARY/ABSTRACT Pathogenic microorganisms are the cause of diverse infections and diseases worldwide. The emergence of drug resistant strains increases the infection and mortality rates and demands new approaches and efforts in the anti-microbial drug discovery. The long-term goal of our research is to understand the role of molecular chaperones in pathogen growth, survival, and virulence and to apply that knowledge in developing molecular approaches to combat infectious diseases. The objective of this application is to develop tools for a high- throughput screen (HTS) for inhibitors of the microbial molecular chaperones from the Hsp100 family. The Hsp100 chaperones reactivate aggregated cellular proteins. A loss of Hsp100 is detrimental for infectivity and survival of a number of bacterial and protozoan pathogens. Importantly, no apparent Hsp100 orthologs are found in metazoan proteomes. This project will lead to the development of a feasible workflow for the future HTS of large compound libraries in search for inhibitors of Hsp100, including the development of methods for hit verification. The following Specific Aims will be pursued: 1. Develop and optimize two HTS assays for the chaperone interactions with substrates and the linkage between the Hsp100 ATPase activity and substrate binding. These assays will use Shigella Hsp100 as the primary target and will be tested for their compatibility with the HTS platform. 2. Perform a pilot screen to test the performance of the assays developed in Aim 1. 3. Evaluate the identified hit compounds in several secondary biochemical and cell-based assays to verify their potency against the Hsp100 activity and to develop tests for elimination of potential false-positive hits. Completion of this project will prepare the stage for the development of Hsp100-targeting drugs through extensive HTS campaigns. This approach is innovative because no chaperone-based antimicrobials have been implemented yet.